Anthrax and other agents of biological warfare have recently received intense publicity. These weapons are an increasingly fearsome danger to our civilization. Agents identified by the CDC (category "A") to pose the greatest threat include Variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), and a group of RNA viruses that cause hemorrhagic fevers (VHFs, e.g., Ebola). Accurate and efficient techniques to identify and diagnose these agents are severely limited. This lack of good diagnostic tests hampers the majority of goals set forth by the NIAID and CDC to prepare the U.S. to counter future bioterrorism attacks. Available older techniques have proven unreliable. Modern molecular tests like individual PCR assays have been developed for some agents. These offer increased speed and sensitivity but because there are so many bioterrorism agents it is prohibitive to run dozens of "singleplex" arrays on each specimen. Similarly, recently reported microchip (MAGI Chip) arrays and other microarrays suffer from either needing PCR amplification first, or from the high cost to make the arrays, and the need for sophisticated equipment. A single assay (or two) that could detect a large number of bioterrorism agents rapidly, sensitively, specifically, and cheaply would greatly enhance antiterrorism planning and biodefense. Our laboratory has pioneered a method of multiplex PCR that can accomplish this goal. This proprietary method (two U.S. patents) has been used commercially in the Hexaplex(r) Assay, which can detect seven common respiratory viruses in a single test. The Specific Aims of this project are: 1) To determine if a multiplex PCR-enzyme hybridization assay (EHA) can be made using our unique technology that will identify all of the CDC Category "A" Bioterrorism agents that are DNA based; 2) RNA based; and finally 3) a single combined multiplex (RNA/DNA) PCR assay with an analytical sensitivity equal to "singleplex" real time assays as developed by the CDC. Specific Aim 4: To determine if this multiplex assay is equivalent to these "singleplex" assays in a clinical trial.